Construction and evaluation of transgenic tobacco plants expressing the coat protein gene of papaya ringspot virus with different translation leaders

Papaya ringspot virus (PRSV) YK isolate used in this study is a local mosai c strain isolated from Yung-Kang, Tainan, and its genome has been cloned an d completely sequenced. A NcoI site before the coat protein (CP) reading fr ame of PRSV YK was generated by oligonucleotide-directed mutagenesis, and t hen the CP reading frame with the 3' noncoding region of PRSV YK was ligate d with the gus leader sequence from the pGEM vector to create the construct pGGCP. To express the CP with a homologous viral translation sequence, the gus leader was replaced by the cDNA sequence corresponding to the 5' regio n (nt 1-347) of PRSV genome to generate a protein containing 9 kDa polypept ide of PRSV P1 protein fused with the CP, and the construct was designated as pG5'CP. In vitro translation from the transcripts derived from pGGCP and pG5'CP generated protein products of 36 kDa and 45 kDa, respectively. Both proteins reacted with the antiserum to PRSV CP, and the level of 36 kDa pr otein was higher than that of 45 kDa protein. The CP reading frame with the gus or PRSV 5' leaders was individually subcloned into a Ti binary vector. Transgenic tobacco plants (Nicotiana tabacum L. Havana 423) expressing the PRSV CP gene with the gus leader (GCP lines) or with the viral leader (5'C P lines) were obtained by Agrobacterium-mediated transformation. When the t ransgenic lines were analyzed by western blotting, the protein products of 36 kDa and 45 kDa reacting to PRSV CP antiserum were detected in the GCP li nes and 5'CP lines, respectively. The presence of the CP gene in the transg enic tobacco was also confirmed by polymerase chain reaction (PCR) using pr imers specific to the CP gene. Analysis of segregation ratios in the R-1 pl ants of four GCP lines and four 5'CP lines indicated that the CP gene in al l of them was nuclearly inherited as a single dominant trait. R-0 and R-1 p lants of the four GCP lines and four 5'CP lines were inoculated with tobacc o etch virus (TEV), potato virus Y (PVY), or pepper mottle virus (PepMoV). The transgenic lines showed significant delay in symptom development and th e severity of symptoms was attenuated. The GCP lines expressing the PRSV CP gene by the gus leader accumulated higher levels of CP and showed higher d egrees of resistance than the 5'CP lines with the PRSV 5' leader. Our resul ts indicate that the homologous viral leader does not enhance CP expression either in vitro or in vivo, nor does it provide better resistance in trans genic tobacco.